The present invention relates to a method of increasing the gene expression of saccharose synthase, to a method of obtaining the saccharose synthase gene, to a new recombinant gene structure, to new vectors containing the recombinant gene structure, to transformed cells containing the recombinant gene structure and to the use of a saccharose synthase to split a dinucleotide.
The saccharose synthase is an enzyme which is limited to plants. In many higher plants there are two different genes known per type; in the case of rice there are three different genes. The differ only slightly in their biochemical characteristics since they are equipped with different promoters and above all control the enzyme expression with precision at different development stages and organs of the plant. Thus in rice 8 days after germation the saccharose synthase 1 is especially located in the roots and stems, while the saccharose synthetic 2 is distributed throughout the entire plant.
Saccharose synthase 3 is especially active in rice grains and thus in a completely different life phase of the plant.
In the plant, the enzyme serves to split saccharose according to the following equation in which, in vivo exclusively the splitting reaction is of significance:
Saccharose+UDP UDP-Glucose+Fructose
The saccharose serves as the transport form of the carbohydrate in plants and thus is split in their target cells, like cells of the storage organs, seeds, developing plant organs inn which the UDP glucose and fructose are directly usable. While the fructose must be initially stored for further use, the UDP glucose is directly available for the synthesis of starch and cellulose. Of economic and scientific interest both the splitting and synthetic directions of the enzyme can be considered. In the synthetic direction, a number of spatially similarly constructed sugars and nonsugars are accepted. In the splitting direction, derivatives of saccharose are accepted and in addition preferably the nucleoside diphosphates (NDPs) UDP, ADP and TDP. Thus a large number of saccharose-analysis disaccharides are available. These are interesting for research into the structure and functioning of glycoconjugates like glycolipids and glycoproteins. Since glycoconjugates also are involved in the communication of cells with one another, many of these structures are also of significance in medicinal research. The enzyme is recoverable from different plant sources. For the processing for example of rice seed, the seed must initially be swelled and then decomposed, the solid component largely filtered off and the filtrate prepurified on an ion exchange column. The large volume resulting from the column step is then reduced and subjected to a gel filtration. The active fractions are suitable for synthesis (compare for this purpose also DE 4 221 595.
The described recovery of saccharose synthase from rice grains is, however, problematical since rice grains have only a limited activity of 0.56 units per gram of dry weight. In addition, the purification is handicapped by the relatively high proportion of carbohydrate in the form of starch and cellulose which is present. Furthermore, the detrimental enzyme invertase, which splits saccharose, cannot be completely separated off. A reduction in the amount of the detrimental enzyme phosphate which decompose NDP""s and the nucleotide sugar splitting enzymes which decompose NDP sugar is desirable. Furthermore, there is the disadvantage that the saccharose synthase in rice apparently is not a pure enzyme but is present in varying proportions of isoenzymes which apparently have different synthetic characteristics. Several syntheses of disaccharides can only be reproduced with difficulty with different enzyme charges. Since the isoenzymes follow different reaction kinetics, measurements of the kinetics are difficult. In addition, the isoenzymes cannot be separated from one another, since they are very similar to one another. Finally, the purification is very slow especially since only small fractions can be charged onto the gel filtration column. A shortening of the purification is thus desirable.
To avoid the aforementioned drawbacks, the method of choice is the use of a recombination microbial system. To be able to produce thereby relatively large quantities of enzyme in an economical and environmentally safe manner, expensive additives or poisons in the cultivation medium must be avoided. Furthermore, only a single saccharose synthase gene can be permitted to be expressed. In the sense of an accelerated purification, expression and/or activity of detrimental enzymes should be held as small as possible. Tests have already been conducted as to the expression of a saccharose synthase gene from Solanum tuberosum, Salanoubat, M. and Belliard, G.: molecular cloning and sequencing of sucrose synthase DNA from potato (Solanum tuberosum L.) preliminary characterization of sucrose synthase mRNA distribution, in: Gene 60, 47-56, 1987) in the Saccharose cerevisiae strain YSH Riesmeier, J. W., et al.: isolation and characterization of a sucrose carrier DNA from spinach by functional expression in yeast. EMBO J 11 (13), 4705-4713, 1992).
For this purpose, the gene is cloned in plasmid 128A2 under control of the ADH promoter (see FIG. 1) and then transformed in the mentioned yeast strain. The ADH promoter normally regulates the reading frequency of the yeast enzyme alcohol dehydrogenase which is constitutively expressed and thus is always provided with exactly equal amounts in the organism. In this manner, the saccharose synthase is expressed with similar expression characteristics under the control of this promoter. This expression results in parts to the yeast strength, indeed the capacity, to split saccharose. However, the specific activity is relatively small: depending upon the method of determination, 5 to 25 mU saccharose synthase/mg of protein can be determined.
It is therefore the object of the invention to provide a method of increasing the gene expression from saccharose synthase by an increased proportion of enzyme. It is further an object of the invention to prepare a composition which can be used in such method.
The objects are achieved by a method in which the expression of the saccharose synthase gene is carried out under the control of a proton-ATPase promoter. The proton-ATPase promoter for this purpose is provided expressly ahead of the saccharose synthase gene. The gene stems preferably from Solanum tuberosum while the protein APTase promoter is preferably from yeast, especially Saccharomyces cerevisiae. A further increase in gene expression is brought about in that the copy number of the saccharose synthase gene and the protein in ATPase promoter is increased. For this purpose, the gene is incorporated with the promoter in a gene construct, preferably in the plasmid pDR195 (compare FIG. 2) and the gene construct is then transformed in a microorganism, especially in the yeast strain Saccharides cerevisiae 22574d (Jauniaux, J.-C. et al. Nitrogen catabolite regulation of proline permease in Saccharomyces cerevisiae. Cloning of the PUT4 gene and study of PUT4RNA levels in wild-type and mutant strains, Eur. J. Biochem, 164, 601-606, 1987).
The recombinant enzyme is preferably purified additionally by means of ion exchange and ultrafiltration after decomposition of the cells. Since it is often required, for protein-chemical application, to prepare very pure protein, the further purification of the technical enzyme preparation can include especially an additional purification step on chelating sepharose.
The saccharose synthase obtained according to the method of the invention can be used for the splitting of disaccharides (for example 2-desoxysaccharose) or N-acetylsaccharosamine with UDP. The enzyme can also be used for the splitting of saccharose with ADP.
The recombinant enzyme is generally usable precisely like the enzyme from rice (see Patent DE 4 221 595). The invention is described in greater detail hereinafter based upon an example.